Standardizing fluorescence microscopy systems

ABSTRACT

Systems and methods for standardizing one or more fluorescence scanning instruments to a reference system by separating the effects of drift and normalization. In an embodiment, a drift image comprising an image of a drift reference slide is captured by a system to be standardized. A drift measurement is calculated using the drift image. A first normalization image comprising an image of a normalization slide is also captured by the system to be standardized. A reference normalization image, also comprising an image of the normalization slide, is captured by a reference system. The first normalization image is compared to the reference normalization image to determine a gamma value and offset value for the system to be standardized.

BACKGROUND

1. Field of the Invention

The present invention is generally related to digital pathology and,more particularly, to correcting the sensitivity of a group of similarfluorescence microscopy imaging instruments, as well maintaining thesensitivity of individual instruments over time and/or environmentalvariations.

2. Related Art

One frequently claimed benefit of fluorescence microscopy is thequantitative nature of signal intensity measurements. Unfortunately,this claimed benefit is commonly misunderstood to imply that fluorescentsignals can easily be related back to some absolute intensity standard.In practice, absolute (calibrated) measurements are difficult, if notimpossible, to achieve due to the lack of stable, calibrated referencestandards.

Fluorescence microscopy system calibration, in terms of absolutesensitivity, requires the use of a calibrated reference standard. Due todifficulties of specimen preparation, and in particular, the unavoidableeffect of sample bleaching over time, it is impossible to create astable, organically based specimen suitable for calibration of afluorescence microscope. Some stable, inorganic specimens are available,but due to variations in spectral response, these are not suitable foraccurate calibration.

Unlike brightfield microscopy, fluorescence signal calibration iscomplicated by the fact that it is not simply a function of broadbandoptical transmission. It also has a strong relationship to wavelength.Fluorochromes have been designed to have very narrow spectral response,with their response curves overlapping the steep edges of multiplebandpass filters within the illumination and imaging paths. Even smallvariation in fluorochrome or filter bandwidths result in changes in thesensitivity of the system. For this reason, it is extremely difficult topredict the overall system sensitivity to a particular fluorochromebased on calibration of individual optical components.

Therefore, what is needed is a system and method that overcomes thesesignificant problems found in conventional systems, as described above.

SUMMARY

The inventors have recognized that in many cases absolute calibration isnot required. Instead, standardizing the response of one or morefluorescence instruments may be sufficient. A better approach is totreat the entire device as a system and to measure the end-to-endresponsiveness of the system using the fluorochrome(s) needing to bestandardized, thereby including the unique spectral characteristics ofboth the sample and the imaging system. However, the difficulty ofsignal drift due to sample fading prevents this method from providing astable reference standard over time. What the inventors have realized isthat standardization can be accomplished by separating the effects ofnormalization and drift.

Other features and advantages of the present invention will become morereadily apparent to those of ordinary skill in the art after reviewingthe following detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The structure and operation of the present invention will be understoodfrom a review of the following detailed description and the accompanyingdrawings in which like reference numerals refer to like parts and inwhich:

FIG. 1 is a block diagram of a first embodiment of an optical microscopysystem according to an embodiment;

FIG. 2 is a block diagram of a second embodiment of an opticalmicroscopy system according to an embodiment;

FIG. 3 is a block diagram of a third embodiment of an optical microscopysystem according to an embodiment;

FIG. 4 is a block diagram illustrating an example set of modules in thescanner system of FIGS. 1-3, according to an embodiment;

FIG. 5 is a block diagram illustrating an example wired or wirelessprocessor-enabled system that may be used in connection with variousembodiments described herein; and

FIG. 6 is a flow diagram illustrating an example process forstandardizing a fluorescence scanning instrument, according to anembodiment.

DETAILED DESCRIPTION

Certain embodiments disclosed herein provide for correcting thesensitivity of a group of similar fluorescence microscopy imaginginstruments, as well as maintaining the sensitivity of individualinstruments over time and/or environmental variations. After readingthis description it will become apparent to one skilled in the art howto implement the invention in various alternative embodiments andalternative applications. However, although various embodiments of thepresent invention will be described herein, it is understood that theseembodiments are presented by way of example only, and not limitation. Assuch, this detailed description of various alternative embodimentsshould not be construed to limit the scope or breadth of the embodimentsof the present invention set forth in the appended claims.

As explained above, the inventors have realized that standardization canbe accomplished by separating the effects of normalization and drift.

The following terms have the following definitions herein:

“Standardization” refers to the general process of making two or moresystems have identical sensitivities. This can be accomplished in a twostep process, comprising normalization and drift correction.

“Normalization” refers to the process of making two or more instrumentsprovide identical results at a particular point in time.

“Drift Correction” refers to the process of making each individualinstrument insensitive to variation over time and/or environmentalconditions.

The inventors have recognized that separation of these two differentaspects of standardization (i.e., normalization and drift correction),enables the use of reference samples that have been optimized for eachtype of correction. Samples using specific fluorochromes enableinclusion of the effects of filter and fluorochrome bandwidth in thecalculations to make one system equivalent to another.

Drift, on the other hand, is mostly insensitive to bandwidth variationsbetween systems, and can be accomplished by using readily availablebroadband reference samples. One example of such samples is coloredplastic microscopy slides. These slides, although not suitable forbandwidth specific standardization, are extremely stable over time, andprovide a broadband spectral response suitable for measuring instrumentsensitivity drift over time.

Image Standardization

In one embodiment, two or more fluorescence microscopy imaginginstruments, A and B, are to be standardized so that intensities derivedfrom each system are equivalent. In this example, A is the referencesystem, and B is the standardized system. The goal is to derive aformula which, when applied to image pixel intensities from system B attime t, will correct each intensity to what would have been obtained onsystem A, at exposure T_(A)(0) at time 0.I _(A)(0)=f[I _(B)(t)]

Variables:

I_(A)(0)=equivalent pixel intensity of System A, at a time 0;

I_(B)(t)=pixel intensity from System B, at a time (t);

G_(B)=Gamma of system B relative to A (constant over time);

O_(B)=Offset of system B relative to A (constant over time);

D_(B)(0)=Drift Factor of system B at time 0;

D_(B)(t)=Drift Factor of system B at time t;

T_(B)(0)=Tissue exposure of system B at time 0; and

T_(B)(t)=Tissue exposure of system B at time t.

Derivation:

For two linear systems, A and B, define a gamma G_(B) and an offsetO_(B) which relate intensity measurements between system B and system Aat time 0 as follows:

$\begin{matrix}{{{IB}(0)} = {\frac{I_{A}(0)}{G_{B}} + {0B}}} & {{equation}\mspace{14mu}(1)}\end{matrix}$

Notably, at time 0, although each system may have acquired images atunique exposures, it is not necessary to explicitly include exposurecorrections in equation (1), because any possible exposure difference isalready included in G and O.

For a particular system, if the sensitivity of that system drifts overtime (e.g., due to light source degradation), then the intensity at timet can be extrapolated from intensity at time 0 by knowing thesensitivity D at time 0 and time t, and correcting for any exposurevariations T.

$\begin{matrix}{{I(t)} = {{I(0)}*\left\lbrack \frac{D(t)}{D(0)} \right\rbrack*\left\lbrack \frac{T(t)}{T(0)} \right\rbrack}} & {{equation}\mspace{14mu}(2)}\end{matrix}$

Using equation (1) to substitute for l (0) in equation (2) gives thefollowing, which relates system B to system A at any time t:

$\begin{matrix}{{{IB}(t)} = {\left( {\left\lbrack \frac{I_{A}(0)}{G_{B}} \right\rbrack + {0B}} \right)*\left\lbrack \frac{D_{B}(t)}{D_{B}(0)} \right\rbrack*\left\lbrack \frac{T_{B}(t)}{T_{B}(0)} \right\rbrack}} & {{equation}\mspace{14mu}(3)}\end{matrix}$

Solving for I_(A)(0) in equation (3) gives the desired relationshipbetween I_(B)(t) and I_(A)(0):

$\begin{matrix}{{{IA}(0)} = {{GB} \cdot \left( {{{{IB}(t)}*\left\lbrack \frac{D_{B}(0)}{D_{B}(t)} \right\rbrack*\left\lbrack \frac{T_{B}(0)}{T_{B}(t)} \right\rbrack} - {0B}} \right)}} & {{equation}\mspace{14mu}(4)}\end{matrix}$

Accordingly, using equation (4) enables standardization of fluorescencemicroscopy systems by determining each of the correction parameters.

Practical Application

FIG. 6 is a flow diagram illustrating an example process forstandardizing a fluorescence scanning instrument, according to anembodiment. In an embodiment, the process begins with capturing theinitial drift reference followed by instrument normalization for eachsystem. A first system is defined as the reference system (System A).All remaining systems (Systems B, C, etc.) are standardized to thereference system.

Step (1) Initial Drift Reference:

A drift reference image is captured using a colored plastic or othersuitable “Drift Slide.” The Drift reference for system A at time 0,D_(A)(0), is calculated per the following:DA(0)=average pixel intensity/exposure  equation (5)

Notably, although each instrument may use a different drift slide, allsubsequent drift captures for each instrument should be performed withthe slide used for its initial drift reference.

Step (1) is repeated for each instrument in turn, calculating initialdrift references for each instrument. Step (2) should be completed assoon as possible in order to minimize any instrument drift between Steps(1) and (2).

Step (2) Normalization:

A slide is selected for the normalization procedure. This slide will beimaged once by each system to be normalized. This specimen should bewell prepared, and representative of the fluorochrome(s) to bestandardized. It should have a broad range of pixel intensities, similarto the samples to be measured. It should also be as stable as possiblewith respect to photobleaching caused by multiple exposures. It does nothave to be stable over long periods of time, as it will not be neededafter the initial instrument normalizations are completed.

An image of the normalization reference slide is captured. This image isused to determine a gamma and offset for each system. The same region ofthe reference slide should be captured for each instrument. Variationsof the region can increase correction inaccuracy.

Step (3) Calculation of Gamma and Offset:

Each system will have an associated gamma and offset, specific to thatsystem. It will not change unless significant changes are made to theillumination or imaging path of the instrument, in which case the entirenormalization procedure should be repeated.

Since system A is the reference system, by definition, Gamma can be setto 1.0 and Offset can be set to 0.

For all remaining systems, the gamma and offset can be derived from theimages of the normalization reference slide. Since there are minorvariations of the images from each machine, due to scale factordifferences or other image warping effects, an image pixel may notexactly overlay the same pixel as captured on another system. These arecalled sub-pixel registration errors. Therefore, it may not be possibleto derive gamma and offset by direct comparison of individual pixelintensities from one system to the corresponding pixel from anothersystem. Instead, in an embodiment, a statistical approach is employedwhich compares the intensity histograms for the two images captured ontwo different systems (e.g., the reference system and the system to bestandardized).

The calculation of the image histograms requires that equivalent areasbe identified in each image. A correlation-based method can be used,whereby a rectangular area in one of the images is chosen as thereference area. A search for an equivalent-sized target area in thesecond image is then made. For each candidate target area, the referenceand target intensity values are compared for each pixel and theroot-mean-squared (RMS) difference is calculated. The target area withthe lowest RMS difference can be chosen as the target area to be used,along with the reference area, in the histogram calculations.

In an embodiment, a “histogram matching” method is used as the basis fordetermining gamma and offset of a system to be standardized from thehistograms. In this method, the cumulative distribution function (CDF)for each image histogram is calculated. A mapping (gamma) is determinedby finding the corresponding intensity in each image for which thecumulative frequencies match.

A drawback with using the gamma function directly is that it may notsample all of the intensity values which will be encountered for futureimages, so that some form of extrapolation is needed. Additionally, thegamma function can have a large number of degrees of freedom (equal tothe number of histogram bins) which may introduce error in the detailsof the gamma function. In an embodiment, these drawbacks can be overcomeby applying a linear regression to the gamma function which reduces thetable of numbers from the gamma mapping to a straight line defined byslope and offset. The slope and offset are the G_(B) and O_(B) inequation (1) above.

Regression also provides an opportunity for estimating errors in thelinear formula. For example, the RMS difference between the straightline and the gamma function is one method for estimating the standarderror in linear prediction. Visual comparison of the regression line tothe gamma function can also reveal any nonlinear bias that may existbetween machines.

Upon completion of step (3), each system will have been normalized withrespect to the reference system, and the initial drift reference of eachsystem will have been determined.

Step (4) Creating Standardized Images:

Depending on the accuracy requirements of the user and the driftcharacteristics of the imaging system, a “Drift Correction” should beperformed at regular intervals for each instrument. In many cases, thiscorrection can be performed once per day, or prior to each use of thesystem. Using the colored plastic slide or other suitable “Drift Slide”from step (1), a drift correction image is captured. The driftcorrection for a system at time t, D(t), is calculated per the followingequation (6):D(t)=average pixel intensity/exposure  equation (6)

Once D(t) has been obtained, the system can be used to capture an imageof any specimen having the same characteristics as used in thenormalization carried out in step (2).

Equation (4) may be used to correct the pixel intensities saved in theimage file, or alternately, the image may be saved unaltered, allowingimage analysis or viewing software to correct pixel intensities as thepixel values are retrieved. Since the resulting image correction canpossibly increase the apparent intensity beyond what the image formatallows, this method has the benefit of maintaining the bestsignal-to-noise ratio while avoiding pixel value “overflow” whichappears as saturation, although the original image pixels were notsaturated.

Benefits of this Approach

In addition to the obvious benefits derived from standardization of twoor more systems, this approach also allows for recovery after loss ofcalibration slides, or damage or intentional modifications to anysystem. It also supports new systems being added to the standardizedset. Once a group of machines have been standardized, the originalreference system is no longer necessary for future standardizationsbecause any standardized system can later be used as a substitute forthe original reference machine. This allows standardization of a machineby comparing the machine to a (virtual) reference machine, therebyreferencing back to the original system.

In one embodiment, a reference machine can be chosen at the factory anda number of standardized machines can be maintained so that any othermachine can always be re-standardized at any point in time in order tomaintain the performance of all machines at all sites within somedesired tolerance.

Example Scanning Instruments

At this point it should be noted that although the various scanninginstruments described herein use line scan cameras to image the sampledata, any type of scanning system that creates a digital image of thesample is suitable for use with the present systems and methods forstandardizing fluorescence microscopy systems.

Turning now to a description of example scanning instruments that couldbe used with the present standardization technique, FIG. 1 illustrates ablock diagram of an embodiment of an optical microscopy system 10. Theheart of the system 10 is a microscope slide scanner 11 that serves toscan and digitize a specimen or sample 12. The sample 12 can be anythingthat may be interrogated by optical microscopy. For instance, the sample12 may be a microscope slide or other sample type that may beinterrogated by optical microscopy. A microscope slide is frequentlyused as a viewing substrate for specimens that include tissues andcells, chromosomes, DNA, protein, blood, bone marrow, urine, bacteria,beads, biopsy materials, or any other type of biological material orsubstance that is either dead or alive, stained or unstained (e.g. usingfluorochromes), labeled or unlabeled. The sample 12 may also be an arrayof any type of DNA or DNA-related material such as cDNA or RNA orprotein that is deposited on any type of slide or other substrate,including any and all samples commonly known as microarrays. The sample12 may be a microtiter plate, for example a 96-well plate. Otherexamples of the sample 12 include integrated circuit boards,electrophoresis records, petri dishes, film, semiconductor materials,forensic materials, or machined parts.

The scanner 11 includes a motorized stage 14, a microscope objectivelens 16, a line scan camera 18, and a data processor 20. The sample 12is positioned on the motorized stage 14 for scanning. The motorizedstage 14 is connected to a stage controller 22 which is connected inturn to the data processor 20. The data processor 20 determines theposition of the sample 12 on the motorized stage 14 via the stagecontroller 22. In the presently preferred embodiment, the motorizedstage 14 moves the sample 12 in at least the two axes (x/y) that are inthe plane of the sample 12. Fine movements of the sample 12 along theoptical z-axis may also be necessary for certain applications of thescanner 11, for example, for focus control. Z-axis movement ispreferably accomplished with a piezo positioner 24, such as the PIFOCfrom Polytec PI or the MIPOS 3 from Piezosystem Jena. The piezopositioner 24 is attached directly to the microscope objective 16 and isconnected to and directed by the data processor 20 via a piezocontroller 27. A means of providing a coarse focus adjustment may alsobe needed and can be provided by z-axis movement as part of themotorized stage 14 or a manual rack-and-pinion coarse focus adjustment(not shown).

In the presently preferred embodiment, the motorized stage 14 includes ahigh precision positioning table with ball bearing linear ways toprovide smooth motion and excellent straight line and flatness accuracy.For example, the motorized stage 14 could include two Daedal model106004 tables stacked one on top of the other. Other types of motorizedstages 14 are also suitable for the scanner 11, including stacked singleaxis stages based on ways other than ball bearings, single- ormultiple-axis positioning stages that are open in the center and areparticularly suitable for trans-illumination from below the sample, orlarger stages that can support a plurality of samples. In the presentlypreferred embodiment, motorized stage 14 includes two stackedsingle-axis positioning tables, each coupled to two millimeterlead-screws and Nema-23 stepping motors. At the maximum lead screw speedof twenty-five revolutions per second, the maximum speed of the sample12 on the motorized stage 14 is fifty millimeters per second. Selectionof a lead screw with larger diameter, for example five millimeters, canincrease the maximum speed to more than 100 millimeters per second. Themotorized stage 14 can be equipped with mechanical or optical positionencoders which has the disadvantage of adding significant expense to thesystem. Consequently, the presently preferred embodiment does notinclude position encoders. However, if one were to use servo motors inplace of stepping motors, then one should use position feedback forproper control.

Position commands from the data processor 20 are converted to motorcurrent or voltage commands in the stage controller 22. In anembodiment, the stage controller 22 includes a 2-axis servo/steppermotor controller (Compumotor 6K2) and two 4-amp microstepping drives(Compumotor OEMZL4). Microstepping provides a means for commanding thestepper motor in much smaller increments than the relatively largesingle 1.8 degree motor step. For example, at a microstep of 100, thesample 12 can be commanded to move at steps as small as 0.1 micrometer.A microstep of 25,000 is used in an embodiment. Smaller step sizes arealso possible. It should be obvious that the optimum selection of themotorized stage 14 and the stage controller 22 depends on many factors,including the nature of the sample 12, the desired time for sampledigitization, and the desired resolution of the resulting digital imageof the sample 12.

The microscope objective lens 16 can be any microscope objective lenscommonly available. One of ordinary skill in the art will realize thatthe choice of which objective lens to use will depend on the particularcircumstances. In an embodiment, the microscope objective lens 16 is ofthe infinity-corrected type.

The sample 12 is illuminated by an illumination system 28 that includesa light source 30 and illumination optics 32. The light source 30 in anembodiment includes a variable intensity halogen light source with aconcave reflective mirror to maximize light output and a KG-1 filter tosuppress heat. However, the light source 30 could also be any other typeof arc-lamp, laser, or other source of light. The illumination optics 32in an embodiment include a standard Köhler illumination system with twoconjugate planes that are orthogonal to the optical axis. Theillumination optics 32 are representative of the bright-fieldillumination optics that can be found on most commercially availablecompound microscopes sold by companies such as Carl Zeiss, Nikon,Olympus, or Leica. One set of conjugate planes includes (i) a field irisaperture illuminated by the light source 30, (ii) the object plane thatis defined by the focal plane of the sample 12, and (iii) the planecontaining the light-responsive elements of the line scan camera 18. Asecond conjugate plane includes (i) the filament of the bulb that ispart of the light source 30, (ii) the aperture of a condenser iris thatsits immediately before the condenser optics that are part of theillumination optics 32, and (iii) the back focal plane of the microscopeobjective lens 16. In an embodiment, the sample 12 is illuminated andimaged in transmission mode, with the line scan camera 18 sensingoptical energy that is transmitted by the sample 12, or conversely,optical energy that is absorbed by the sample 12.

The scanner 11 is equally suitable for detecting optical energy that isreflected from the sample 12, in which case the light source 30, theillumination optics 32, and the microscope objective lens 16 must beselected based on compatibility with reflection imaging. One possibleembodiment may therefore be illumination through a fiber optic bundlethat is positioned above or at an angle to the sample 12. Otherpossibilities include excitation that is spectrally conditioned by amonochromator. If the microscope objective lens 16 is selected to becompatible with phase-contrast microscopy, then the incorporation of atleast one phase stop in the condenser optics that are part of theillumination optics 32 will enable the scanner 11 to be used for phasecontrast microscopy. To one of ordinary skill in the art, themodifications required for other types of microscopy such asdifferential interference contrast and confocal microscopy should bereadily apparent. Overall, the scanner 11 is suitable, with appropriatebut well-known modifications, for the interrogation of microscopicsamples in any known mode of optical microscopy.

Between the microscope objective lens 16 and the line scan camera 18 aresituated the line scan camera focusing optics 34 that focus the opticalsignal captured by the microscope objective lens 16 onto thelight-responsive elements of the line scan camera 18. In a moderninfinity-corrected microscope the focusing optics between the microscopeobjective lens and the eyepiece optics, or between the microscopeobjective lens and an external imaging port, consist of an opticalelement known as a tube lens that is part of a microscope's observationtube. Many times the tube lens consists of multiple optical elements toprevent the introduction of coma or astigmatism. One of the motivationsfor the relatively recent change from traditional finite tube lengthoptics to infinity corrected optics was to increase the physical spacein which the optical energy from the sample 12 is parallel, meaning thatthe focal point of this optical energy is at infinity. In this case,accessory elements like dichroic mirrors or filters can be inserted intothe infinity space without changing the optical path magnification orintroducing undesirable optical artifacts.

Infinity-corrected microscope objective lenses are typically inscribedwith an infinity mark. The magnification of an infinity correctedmicroscope objective lens is given by the quotient of the focal lengthof the tube lens divided by the focal length of the objective lens. Forexample, a tube lens with a focal length of 180 millimeters will resultin 20× magnification if an objective lens with 9 millimeter focal lengthis used. One of the reasons that the objective lenses manufactured bydifferent microscope manufacturers are not compatible is because of alack of standardization in the tube lens focal length. For example, a20× objective lens from Olympus, a company that uses a 180 millimetertube lens focal length, will not provide a 20× magnification on a Nikonmicroscope that is based on a different tube length focal length of 200millimeters. Instead, the effective magnification of such an Olympusobjective lens engraved with 20× and having a 9 millimeter focal lengthwill be 22.2×, obtained by dividing the 200 millimeter tube lens focallength by the 9 millimeter focal length of the objective lens. Changingthe tube lens on a conventional microscope is virtually impossiblewithout disassembling the microscope. The tube lens is part of acritical fixed element of the microscope. Another contributing factor tothe incompatibility between the objective lenses and microscopesmanufactured by different manufacturers is the design of the eyepieceoptics, the binoculars through which the specimen is observed. Whilemost of the optical corrections have been designed into the microscopeobjective lens, most microscope users remain convinced that there issome benefit in matching one manufacturer's binocular optics with thatsame manufacturer's microscope objective lenses to achieve the bestvisual image.

The line scan camera focusing optics 34 include a tube lens opticmounted inside of a mechanical tube. Since the scanner 11, in itspreferred embodiment, lacks binoculars or eyepieces for traditionalvisual observation, the problem suffered by conventional microscopes ofpotential incompatibility between objective lenses and binoculars isimmediately eliminated. One of ordinary skill will similarly realizethat the problem of achieving parfocality between the eyepieces of themicroscope and a digital image on a display monitor is also eliminatedby virtue of not having any eyepieces. Since the scanner 11 alsoovercomes the field of view limitation of a traditional microscope byproviding a field of view that is practically limited only by thephysical boundaries of the sample 12, the importance of magnification inan all-digital imaging microscope such as provided by the presentscanner 11 is limited. Once a portion of the sample 12 has beendigitized, it is straightforward to apply electronic magnification,sometimes known as electric zoom, to an image of the sample 12 in orderto increase its magnification. Increasing the magnification of an imageelectronically has the effect of increasing the size of that image onthe monitor that is used to display the image. If too much electroniczoom is applied, then the display monitor will be able to show onlyportions of the magnified image. It is not possible, however, to useelectronic magnification to display information that was not present inthe original optical signal that was digitized in the first place. Sinceone of the objectives of the scanner 11 is to provide high qualitydigital images, in lieu of visual observation through the eyepieces of amicroscope, it is important that the content of the images acquired bythe scanner 11 include as much image detail as possible. The termresolution is typically used to describe such image detail and the termdiffraction-limited is used to describe the wavelength-limited maximumspatial detail available in an optical signal. The scanner 11 providesdiffraction-limited digital imaging by selection of a tube lens focallength that is matched according to the well know Nyquist samplingcriteria to both the size of an individual pixel element in alight-sensing camera such as the line scan camera 18 and to thenumerical aperture of the microscope objective lens 16. It is well knownthat numerical aperture, not magnification, is the resolution-limitingattribute of a microscope objective lens 16.

An example will help to illustrate the optimum selection of a tube lensfocal length that is part of the line scan camera focusing optics 34.Consider again the 20× microscope objective lens 16 with 9 millimeterfocal length discussed previously and assume that this objective lenshas a numerical aperture of 0.50. Assuming no appreciable degradationfrom the condenser, the diffraction-limited resolving power of thisobjective lens at a wavelength of 500 nanometers is approximately 0.6micrometers, obtained using the well-known Abbe relationship. Assumefurther that the line scan camera 18, which in an embodiment has aplurality of 14 micrometer square pixels, is used to detect a portion ofthe sample 12. In accordance with sampling theory, it is necessary thatat least two sensor pixels subtend the smallest resolvable spatialfeature. In this case, the tube lens must be selected to achieve amagnification of 46.7, obtained by dividing 28 micrometers, whichcorresponds to two 14 micrometer pixels, by 0.6 micrometers, thesmallest resolvable feature dimension. The optimum tube lens optic focallength is therefore about 420 millimeters, obtained by multiplying 46.7by 9. The line scan focusing optics 34 with a tube lens optic having afocal length of 420 millimeters will therefore be capable of acquiringimages with the best possible spatial resolution, similar to what wouldbe observed by viewing a specimen under a microscope using the same 20×objective lens. To reiterate, the scanner 11 utilizes a traditional 20×microscope objective lens 16 in a higher magnification opticalconfiguration, in this example about 47×, in order to acquirediffraction-limited digital images. If a traditional 20× magnificationobjective lens 16 with a higher numerical aperture were used, say 0.75,the required tube lens optic magnification for diffraction-limitedimaging would be about 615 millimeters, corresponding to an overalloptical magnification of 68×. Similarly, if the numerical aperture ofthe 20× objective lens were only 0.3, the optimum tube lens opticmagnification would only be about 28×, which corresponds to a tube lensoptic focal length of approximately 252 millimeters. The line scancamera focusing optics 34 may be modular elements of the scanner 11which can be interchanged as necessary for optimum digital imaging. Theadvantage of diffraction-limited digital imaging is particularlysignificant for applications, for example bright field microscopy, inwhich the reduction in signal brightness that accompanies increases inmagnification is readily compensated by increasing the intensity of anappropriately designed illumination system 28.

In principle, it is possible to attach external magnification-increasingoptics to a conventional microscope-based digital imaging system toeffectively increase the tube lens magnification so as to achievediffraction-limited imaging as has just been described for the presentscanner 11. However, the resulting decrease in the field of view isoften unacceptable, making this approach impractical. Furthermore, manyusers of microscopes typically do not understand enough about thedetails of diffraction-limited imaging to effectively employ thesetechniques on their own. In practice, digital cameras are attached tomicroscope ports with magnification-decreasing optical couplers toattempt to increase the size of the field of view to something moresimilar to what can be seen through the eyepiece. The standard practiceof adding de-magnifying optics is a step in the wrong direction if thegoal is to obtain diffraction-limited digital images.

In a conventional microscope, different power objectives lenses aretypically used to view the specimen at different resolutions andmagnifications. Standard microscopes have a nosepiece that holds fiveobjectives lenses. In an all-digital imaging system, such as the presentscanner 11, there is a need for only one microscope objective lens 16with a numerical aperture corresponding to the highest spatialresolution desirable. An embodiment of the scanner 11 provides for onlyone microscope objective lens 16. Once a diffraction-limited digitalimage has been captured at this resolution, it is straightforward usingstandard digital image processing techniques, to present imageryinformation at any desirable reduced resolutions and magnifications.

In an embodiment, the scanner 11 is based on a Dalsa SPARK line scancamera 18 with 1024 pixels (picture elements) arranged in a lineararray, with each pixel having a dimension of 14 by 14 micrometers. Anyother type of linear array, whether packaged as part of a camera orcustom-integrated into an imaging electronic module, can also be used.The linear array in the an embodiment effectively provides eight bits ofquantization, but other arrays providing higher or lower level ofquantization may also be used. Alternate arrays based on 3-channelred-green-blue (RGB) color information or time delay integration (TDI),may also be used. TDI arrays provide a substantially bettersignal-to-noise ratio (SNR) in the output signal by summing intensitydata from previously imaged regions of a specimen, yielding an increasein the SNR that is in proportion to the square-root of the number ofintegration stages. TDI arrays can comprise multiple stages of lineararrays. TDI arrays are available with 24, 32, 48, 64, 96, or even morestages. The scanner 11 can also support linear arrays that aremanufactured in a variety of formats including some with 512 pixels,some with 1024 pixels, and others having as many as 4096 pixels.Appropriate, but well known, modifications to the illumination system 28and the line scan camera focusing optics 34 may be required toaccommodate larger arrays. Linear arrays with a variety of pixel sizescan also be used in scanner 11. The salient requirement for theselection of any type of line scan camera 18 is that the sample 12 canbe in motion with respect to the line scan camera 18 during thedigitization of the sample 12 in order to obtain high quality images,overcoming the static requirements of the conventional imaging tilingapproaches known in the prior art.

The output signal of the line scan camera 18 is connected to the dataprocessor 20. The data processor 20 in an embodiment includes a centralprocessing unit with ancillary electronics, for example a motherboard,to support at least one signal digitizing electronics board such as animaging board or a frame grabber. In an embodiment, the imaging board isan EPIX PIXCID24 PCI bus imaging board. However, there are many othertypes of imaging boards or frame grabbers from a variety ofmanufacturers which could be used in place of the EPIX board. Analternate embodiment could be a line scan camera that uses an interfacesuch as IEEE 1394, also known as Firewire, to bypass the imaging boardaltogether and store data directly on a data storage 38, such as a harddisk.

The data processor 20 is also connected to a memory 36, such as randomaccess memory (RAM), for the short-term storage of data, and to the datastorage 38, such as a hard drive, for long-term data storage. Further,the data processor 20 is connected to a communications port 40 that isconnected to a network 42 such as a local area network (LAN), a widearea network (WAN), a metropolitan area network (MAN), an intranet, anextranet, or the global Internet. The memory 36 and the data storage 38are also connected to each other. The data processor 20 is also capableof executing computer programs, in the form of software, to controlcritical elements of the scanner 11 such as the line scan camera 18 andthe stage controller 22, or for a variety of image-processing functions,image-analysis functions, or networking. The data processor 20 can bebased on any operating system, including operating systems such asWindows, Linux, OS/2, Mac OS, and Unix. In the presently preferredembodiment, the data processor 20 operates based on the Windows NToperating system.

The data processor 20, memory 36, data storage 38, and communicationport 40 are each elements that can be found in a conventional computer.One example would be a personal computer such as a Dell Dimension XPST500 that features a Pentium III 500 MHz processor and up to 756megabytes (MB) of RAM. In the presently preferred embodiment, thecomputer, elements which include the data processor 20, memory 36, datastorage 38, and communications port 40 are all internal to the scanner11, so that the only connection of the scanner 11 to the other elementsof the system 10 is the communication port 40. In an alternateembodiment of the scanner 11, the computer elements would be external tothe scanner 11 with a corresponding connection between the computerelements and the scanner 11.

The scanner 11, in the presently preferred embodiment of the invention,integrates optical microscopy, digital imaging, motorized samplepositioning, computing, and network-based communications into asingle-enclosure unit. An advantage of packaging the scanner 11 as asingle-enclosure unit with the communications port 40 as the primarymeans of data input and output are reduced complexity and increasedreliability. The various elements of the scanner 11 are optimized towork together, in sharp contrast to traditional microscope-based imagingsystems in which the microscope, light source, motorized stage, camera,and computer are typically provided by different vendors and requiresubstantial integration and maintenance.

The communication port 40 provides a means for rapid communications withthe other elements of the system 10, including the network 42. Anexample communications protocol for the communications port 40 is acarrier-sense multiple-access collision detection protocol such asEthernet, together with the TCP/IP protocol for transmission control andinternetworking. The scanner 11 is intended to work with any type oftransmission media, including broadband, baseband, coaxial cable,twisted pair, fiber optics, DSL, or wireless.

In an embodiment, control of the scanner 11 and review of the imagerydata captured by the scanner 11 are performed on a computer 44 that isconnected to the network 42. The computer 44, in its presently preferredembodiment, is connected to a display monitor 46 to provide imageryinformation to an operator. A plurality of computers 44 may be connectedto the network 42. In an embodiment, the computer 44 communicates withthe scanner 11 using a network browser such as Internet Explorer fromMicrosoft or Netscape Communicator from AOL. Images can be stored on thescanner 11 in a common compressed format such a JPEG which is an imageformat that is compatible with standard image-decompression methods thatare already built into most commercial browsers. Other standard ornon-standard, lossy or lossless, image compression formats will alsowork. In the presently preferred embodiment, the scanner 11 is a webserver providing an operator interface that is based on web pages thatare sent from the scanner 11 to the computer 44. For dynamic review ofimagery data, an embodiment of the scanner 11 is based on playing back,for review on the display monitor 46 that is connected to the computer44, multiple frames of imagery data using standard multiple-framebrowser compatible software packages such as Media-Player fromMicrosoft, Quicktime from Apple Computer, or RealPlayer from RealNetworks. In the presently preferred embodiment, the browser on thecomputer 44 uses the hypertext transmission protocol (HTTP) togetherwith TCP for transmission control.

There are, and will be in the future, many different means and protocolsby which the scanner 11 could communicate with the computer 44, or aplurality of computers. While the some embodiments are based on standardmeans and protocols, the approach of developing one or multiplecustomized software modules known as applets is equally feasible and maybe desirable for selected future applications of the scanner 11.Further, there are no constraints that computer 44 be of any specifictype such as a personal computer (PC) or be manufactured by any specificcompany such as Dell. One of the advantages of a standardizedcommunications port 40 is that any type of computer 44 operating commonnetwork browser software can communicate with the scanner 11.

If one so desires, it is possible, with some modifications to thescanner 11, to obtain spectrally resolved images. Spectrally resolvedimages are images in which spectral information is measured at everyimage pixel. Spectrally resolved images could be obtained by replacingthe line scan camera 18 of the scanner 11 with an optical slit and animaging spectrograph. The imaging spectrograph uses a two-dimensionalCCD detector to capture wavelength-specific intensity data for a columnof image pixels by using a prism or grating to disperse the opticalsignal that is focused on the optical slit along each of the rows of thedetector.

FIG. 2 illustrates a block diagram of a second embodiment of an opticalmicroscopy system 10, according to an embodiment. In this system 10, thescanner 11 is more complex and expensive than the embodiment shown inFIG. 1. The additional attributes of the scanner 11 that are shown donot all have to be present for any alternate embodiment to functioncorrectly. FIG. 2 is intended to provide a reasonable example ofadditional features and capabilities that could be incorporated into thescanner 11.

The alternate embodiment of FIG. 2 can provide for a much greater levelof automation than the presently preferred embodiment of FIG. 1. A morecomplete level of automation of the illumination system 28 is achievedby connections between the data processor 20 and both the light source30 and the illumination optics 32 of the illumination system 28. Theconnection to the light source 30 may control the voltage, or current,in an open or closed loop fashion, in order to control the intensity ofthe light source 30. Recall that the light source 30 is a halogen bulbin the presently preferred embodiment. The connection between the dataprocessor 20 and the illumination optics 32 could provide closed loopcontrol of the field iris aperture and the condenser iris to provide ameans for ensuring that optimum Köhler illumination is maintained.

Use of the scanner 11 for fluorescence imaging requires easilyrecognized modifications to the light source 30, the illumination optics32, and the microscope objective lens 16. The second embodiment of FIG.2 also provides for a fluorescence filter cube 50 that includes anexcitation filter, a dichroic filter, and a barrier filter. Thefluorescence filter cube 50 is positioned in the infinity corrected beampath that exists between the microscope objective lens 16 and line scancamera focusing optics 34. One embodiment for fluorescence imaging couldinclude the addition of a filter wheel or tunable filter into theillumination optics 32 to provide appropriate spectral excitation forthe variety of fluorescent dyes or nano-crystals available on themarket.

The addition of at least one beam splitter 52 into the imaging pathallows the optical signal to be split into at least two paths. Theprimary path is via the line scan camera focusing optics 34, asdiscussed previously, to enable diffraction-limited imaging by the linescan camera 18. A second path is provided via an area scan camerafocusing optics 54 for imaging by an area scan camera 56. It should bereadily apparent that proper selection of these two focusing optics canensure diffraction-limited imaging by the two camera sensors havingdifferent pixel sizes. The area scan camera 56 can be one of many typesthat are currently available, including a simple color video camera, ahigh performance, cooled, CCD camera, or a variable integration-timefast frame camera. The area scan camera 56 provides a traditionalimaging system configuration for the scanner 11. The area scan camera 56is connected to the data processor 20. If two cameras are used, forexample the line scan camera 18 and the area scan camera 56, both cameratypes could be connected to the data processor using either a singledual-purpose imaging board, two different imaging boards, or theIEEE1394 Firewire interface, in which case one or both imaging boardsmay not be needed. Other related methods of interfacing imaging sensorsto the data processor 20 are also available.

While the primary interface of the scanner 11 to the computer 44 is viathe network 42, there may be instances, for example a failure of thenetwork 42, where it is beneficial to be able to connect the scanner 11directly to a local output device such as a display monitor 58 and toalso provide local input devices such as a keyboard and mouse 60 thatare connected directly to the data processor 20 of the scanner 11. Inthis instance, the appropriate driver software and hardware should beprovided as well.

The second embodiment shown in FIG. 2 can also provide for a muchgreater level of automated imaging performance. Enhanced automation ofthe imaging of the scanner 11 can be achieved by closing the focuscontrol loop comprising the piezo positioner 24, the piezo controller26, and the data processor 20 using well-known methods of autofocus. Thesecond embodiment also provides for a motorized nose-piece 62 toaccommodate several objectives lenses. The motorized nose-piece 62 isconnected to and directed by the data processor 20 through a nose-piececontroller 64.

There are other features and capabilities of the scanner 11 which couldbe incorporated. For example, the process of scanning (i.e., moving) thesample 12 with respect to the microscope objective lens 16 that issubstantially stationary in the x/y plane of the sample 12 could bemodified to comprise scanning (i.e., moving) the microscope objectivelens 16 with respect to a stationary sample 12. Scanning the sample 12,or scanning the microscope objective lens 16, or scanning both thesample 12 and the microscope objective lens 16 simultaneously, arepossible embodiments of the scanner 11 which can provide the same largecontiguous digital image of the sample 12 as discussed previously.

The scanner 11 also provides a general purpose platform for automatingmany types of microscope-based analyses. The illumination system 28could be modified from a traditional halogen lamp or arc-lamp to alaser-based illumination system to permit scanning of the sample 12 withlaser excitation. Modifications, including the incorporation of aphotomultiplier tube or other non-imaging detector, in addition to or inlieu of the line scan camera 18 or the area scan camera 56, could beused to provide a means of detecting the optical signal resulting fromthe interaction of the laser energy with the sample 12.

FIG. 3 is a block diagram of a third embodiment of an optical microscopysystem 10 according to an embodiment. In the illustrated embodiment, thefluorescence scanner system 11 comprises a processor 245 that iscommunicatively coupled with a data storage area 38 that can include,e.g., volatile and persistent computer readable storage mediums. Theprocessor 245 executes programmed modules in memory 36 and/or datastorage 38 to control the macro camera 240, TDI line scan camera 205,focusing optics 210, motorized filter cube turret 215, and objectivepositioner 220 that is coupled to the objective lens 225. The processoralso executes programmed modules to control the illumination module 235,motion controller 250 and motorized stage 255 that supports the sample230. The processor also executes programmed modules to control the lightsource 265, the optimized epifluorescence illumination module 290 thatcomprises the epifluorescence illumination optics 270 and the optionalbeam shaping optics 275, and the motorized excitation filter wheel 280.

In operation, the various components of the fluorescence scanner system11 and the programmed modules stored in memory 36 and/or data storage 38enable automatic scanning and digitizing of the fluorescence sample 230.Microscope slides (not shown) are often used as a platform to supportthe fluorescence sample 230 and can be securely placed on the motorizedstage 255 of the fluorescence scanner system 11 for scanning the sample230. Under control of the processor 245, the motorized stage 255accelerates the sample 230 to a substantially constant velocity forsensing by the TDI line scan camera 205, where the speed of the stage issynchronized with the line rate of the TDI line scan camera 205. Afterscanning of a stripe of image data, the motorized stage 255 deceleratesand brings the sample 230 to a substantially complete stop beforeadditional scanning of the same stripe or a different stripe.

The sample 230 can be any type of specimen that has been labeled withflorescence dyes or fluorochrome, for example, tissue, cells, DNA andprotein are types of samples, just to name a few. The fluorescencesample 230 can also be an array of specimen, for example, tissuesections, DNA, or DNA related material deposited on a substrate. As willbe understood by those skilled in the art, any fluorescence specimenthat can be interrogated by a fluorescence optical microscope can alsobe scanned by the fluorescence scanner system 11 to create a digitalslide image of the fluorescence sample 230.

Fluorescence molecules are photon sensitive molecules that can absorblight at a specific wavelength (excitation). These photon sensitivemolecules also emit light at a higher wavelength (emission). Because theefficiency of this photoluminescence phenomenon is very low, the amountof emitted light is often very low. The low amount of emitted lightfrustrates conventional techniques for scanning and digitizing thesample 230. Advantageously, use of the TDI line scan camera 205 thatincludes multiple linear sensor arrays increases the sensitivity of thecamera by exposing the same area of the sample 230 to the multiplelinear sensor arrays of the TDI line scan camera 205. This isparticularly useful when scanning faint fluorescence samples with lowemitted light. In alternative embodiments, the TDI line scan camera 205may include 64, 96 or 120 linear sensor arrays, which may be chargecoupled device (“CCD”) arrays.

In one embodiment, the TDI line scan camera 205 is a monochrome TDI linescan camera, although the systems and methods described herein are notlimited to monochrome cameras. Advantageously, monochrome images areideal in fluorescence microscopy because they provide a more accuraterepresentation of the actual signals from the various channels presenton the sample. As will be understood by those skilled in the art, afluorescence sample 230 can be labeled with multiple florescence dyesthat emit light at different wavelengths, which are also referred to as“channels.”

Furthermore, because the low and high end signal levels of variousfluorescence samples present a wide spectrum of wavelengths for the TDIline scan camera 205 to sense, it is desirable for the low and high endsignal levels that the TDI line scan camera 205 can sense to besimilarly wide. Accordingly, in one embodiment a TDI line scan camera205 used in the fluorescence scanning system 11 is a monochrome 10-bit64 stage TDI line scan camera. It should be noted that a variety of bitdepths for the TDI line scan camera 205 can be employed for use with thefluorescence scanning system 11.

In one embodiment, the fluorescence scanning system 11 uses a highprecision and tightly coordinated XY grid to aid in the location of thesample 230 on the motorized stage 255. In one embodiment, the motorizedstage 255 is a linear motor based XY stage with high precision encodersemployed on both the X and the Y axis. For example, a 50 nanometerencoder can be used on the axis in the scanning direction and a 5nanometer encoder can be used on the axis that is in the directionperpendicular to the scanning direction and on the same plane. Objectivelens 225 is also mounted on the objective positioner 220 which employs alinear motor on the optical axis with a 50 nanometer encoder. In oneembodiment, the three XYZ axes are coordinated and controlled in aclosed loop manner using motion controller 250 that includes a motioncontrol drive (not shown) and a motion control board (not shown).Control and coordination is maintained by processor 245 that employsmemory a36 and/or data storage area 38 for storing information andinstructions, including the computer executable programmed steps forscanning system 11 operation.

In one embodiment, the objective lens 225 is a plan APO infinitycorrected objective that is suitable for fluorescence microscopy (e.g.,an Olympus 20×, 0.75 NA). Advantageously, objective lens 225 is capableof correcting for chromatic and spherical aberrations. Because objectivelens 225 is infinity corrected, other optical components such asfilters, magnification changer lenses, etc. can be placed in the opticalpath above the objective lens 225 where the light beam passing throughthe objective lens becomes a collimated light beam. The objective lens255 combined with focusing optics 210 provides the total magnificationfor the fluorescence scanning system 11 and also provides for focusingon the surface of TDI line scan camera 205. The focusing optics 210contain a tube lens and an optional 2× magnification changer. In oneembodiment, the 2× magnification changer can allow objective lens 255that is natively 20× to scan a sample 230 at 40× magnification.

For the scanning system 11 to effectively perform fluorescencemicroscopy, a suitable light source 265 needs to be employed. Inalternative embodiments, arc lamps such as mercury, metal halide, xenonlamps or LED light sources can be used for this purpose. In oneembodiment, light source 265 is an arc based light source such as a 200watt mercury based DC operated and processor controlled light source.Advantageously, the light source 265 allows the processor 245 to manageshutter control and iris control. In one embodiment, a liquid lightguide (not shown) can be used to deliver light to the field of view ofthe objective lens 225, where scanning takes place, or to otherdesirable locations within the fluorescence scanning system 11. Forexample, a 3 mm core liquid light guide can be used to deliver light.

The fluorescence scanning system 11 additionally includes illuminationoptics that include epifluorescence illumination optics 270 and optionalbeam shaping optics 275 that collectively are shown as optimizedepifluorescence illumination 290. The epifluorescence illuminationoptics 270 condenses the excitation light on the sample 230 through theobjective lens 225. As is the case in epifluorescence illumination, theemitted light from the sample is also collected with the same objectivelens 225. One particular advantage of using epifluorescence illuminationis to maximize the blockage of excitation light reaching the multiplelinear array sensors of the TDI line scan camera 205. In turn, this alsomaximizes the amount of emitted light that reaches the multiple lineararray sensors of the TDI line scan camera 205.

The epifluorescence illumination optics 270 can be implemented usingDichroic mirrors that provide wavelength dependent reflectivity andtransmission. As a result, the excitation light gets reflected off thesurface of a Dichroic mirror and is guided through the objective lens225 to reach the sample 230. However, the emitted light from the sample230, which is at a higher wavelength, will pass through the Dichroicmirror and reach the multiple linear array sensors of the TDI line scancamera 205.

The epifluorescence illumination optics 270 also collimates the lightfrom the light source 265. In alternative embodiments, this isaccomplished using Kohler or Critical illumination. Kohler illuminationprovides the most uniform light illumination on the sample 230 tominimize shading in digital slide images while Critical illuminationprovides the maximum light intensity on the sample to decrease thenecessary imaging exposure time. Both Kohler and Critical illuminationcan be employed by the fluorescence scanning system 11.

In one embodiment, the epifluorescence illumination optics 270 includerelay lens tube optics (not shown) that are designed to collimate thelight received from light source 265 through a liquid light guide (notshown) and deliver the light to the sample 230. In this embodiment, thelight profile on the sample has minimal roll off within the imaging viewthrough the objective lens 225.

In an alternative embodiment, the optional beam shaping optics 275operate to illuminate just the portion of the sample 235 that is beingsensed by the multiple linear array sensors of the TDI line scan camera205. Advantageously, the beam shaping optics 275 reshape theillumination area from its natural circular shape into a thin oval shapethat closely approximates the rectangular sensor area. The reshapedillumination area advantageously also receives increased light energy.Reshaping the illumination area is a vast improvement over conventionalmasking, which discards all of the light energy outside of therectangular mask. Advantages of the optional beam shaping optics 275include: (a) preserving the sample from redundant exposure to theexcitation light and thereby minimizing photobleaching of the sample;and (b) increasing the light energy delivered to the illumination areaon the sample and thereby allowing shorter exposure times (i.e., higherline rates) during scanning of the sample 230 to create a digital slideimage. In combination, these two can provide a significant advantage.

The fluorescence scanning system 11 also includes motorized excitationfilter wheel 280 that facilitates configuration and use of variousfilters. It is desirable to have certain filters available to increasethe effectiveness of fluorescence microscopy. Some of the desirablefilters include: (a) an excitation filter that narrows down the broadband light generated from the light source 265 to the specific bandneeded for excitation of the sample 230; (b) an emission filter tofilter out excess light and possibly excitation light that may reach oneor more of the linear array sensors of the TDI line scan camera 205; and(c) a Dichroic mirror as described above for use with epi-fluorescenceillumination. Other filters can also be included.

In one embodiment, the fluorescence scanning system 11 includes amotorized wheel for excitation filters, a motorized wheel for emissionfilters, and a motorized wheel for Dichroic mirrors. Sliders can also beused in place of wheels. In an alternative embodiment, the fluorescencescanning system 11 includes a motorized excitation filter wheel 280 anda motorized filter cube turret 215 that includes the emission filter andthe Dichroic mirrors. One particular advantage of separating theexcitation filter(s) from the emission filter(s) and the dichroicmirrors is related to the use of a TDI line scan camera 205 that ismonochrome. Specifically, use of a monochrome TDI line scan camera 205causes each stripe region of the sample 230 to be scanned and digitizedmultiple times—once for each emission wavelength (i.e., channel) to beinterrogated. Registration of the multiple scans of a single stripe istherefore critically important to allow integration of the multiplescans into a single image that includes information from each channel.

Additionally, because the motorized filter cube turret 215 includes anemission filter and the Dichroic mirrors, the motorized filter cubeturret 215 can implement a filter configuration in which multiple bandfilter cubes (Dichroic mirrors and emission filters) and single bandexcitation filters are used in combination (called a “Pinkel”configuration). Use of the Pinkel configuration advantageously allowsscanning and digitization of the sample 230 multiple times whilechanging only the excitation filter using the motorized excitationfilter wheel 280. Consequently, no mechanical or optical registrationissues will be observed between images of the same stripe on the samplebecause there is no moving component in the imaging path. This is asignificant advantage of separating the excitation filter(s) from theemission filter(s) and Dichroic mirrors when using a line scan camera(such as TDI line scan camera 205) that scans the same area of thesample 230 multiple times and combines the resulting multiple images.

In one embodiment, motorized excitation filter wheel 280 is a sixposition wheel that can accommodate standard 25 mm filters. Themotorized excitation filter wheel 280 is under the control of theprocessor 245. As will be understood by those skilled in the art, anystandard fluorescence filter can be used. Preferably, hard coatedfilters are used with the scanning system 11 because they are moredurable and are easy to clean.

In one embodiment, motorized filter cube turret 215 is also a sixpositioned turret that holds filter cubes, for example standard Olympusfilter cubes can be used. The motorized filter cube turret 215 is underthe control of the processor 245. Both the motorized excitation filterwheel 280 and the motorized filter cube turret 215 filter areautomatically placed in the illumination path or imaging path undercontrol of the processor 245 depending on the particular fluorochromeson the sample 230 and the available filters configured in thefluorescence scanning system 11.

One particular challenge when imaging fluorescence samples isrecognizing the sample 230 on the microscope slide and determining thearea to be scanned and digitized into a digital slide image. Moreover,fluorescence samples often times appear to be transparent, whichamplifies this challenge because regular imaging of the fluorescencesample using regular lighting does not necessarily provide a means torecognize the specimen on the slide. Accordingly, illumination module235 is configured to apply oblique illumination to the fluorescencesample 230. Macro camera 240 is configured to capture the image of thesample after it is illuminated with oblique lighting by the illuminationmodule 235. Advantageously, the tissue in the resulting image has enoughcontrast to be recognizable.

In one embodiment, illumination module 235 uses a white LED module (notshown) integrated with a beam diffuser lens (also not shown) positionedat an angle to illuminate the sample 230 at an appropriate angle. In oneembodiment, oblique illumination at an angle of 30 degrees is used (asmeasured with respect to the surface of the microscope slide). However,it should be understood that oblique illumination at any angle in therange of 0 degrees to 85 degrees can be used. A zero degree angle (e.g.,illuminating the sample 230 through the thickness of the slide) can beaccomplished using, for example, a linear fiber. In one embodiment,additional shrouding around the illumination module 235 is provided tobetter channel the oblique light hitting the surface of the sample 230.Shrouding around the macro camera 240 is also provided to increase theamount of reflected light off the sample 230 that is captured by themacro camera 240 while also minimizing the amount of illumination lightthat is captured by the macro camera 240.

The processor 245 may include one or more processor cores, as will beunderstood by those skilled in the art. Additional separate processorsmay also be provided to control particular components or performparticular functions. For example, additional processors may include anauxiliary processor to manage data input, an auxiliary processor toperform floating point mathematical operations, a special-purposeprocessor having an architecture suitable for fast execution of signalprocessing algorithms (e.g., digital signal processor), a slaveprocessor subordinate to the main processor (e.g., back-end processor),an additional processor for controlling the TDI line scan camera 205,the stage 255, the objective lens 225 or a display (not shown). Suchadditional processors may be separate discrete processors or may beintegrated with the processor 245.

The processor 245 is preferably electrically connected to the variouscomponents of the fluorescence scanning system 11 in order to providecontrol and coordination and overall management of the fluorescencescanning system 11. Alternatively, the processor 245 can be in wirelesscommunication with one or more of the various components of thefluorescence scanning system 11.

The memory 36 and data storage area 38 provide storage of data andinstructions for programs executing on the processor 245. The memory 36and/or data storage area 38 can include volatile and persistent storageof the data and instructions and may include a random access memory, aread only memory, a hard disk drive, removable storage drive, and thelike.

The scanning system 11 may also include a communication interface (notshown) that allows software and data to be transferred between thescanning system 11 and external devices that are directly connected(e.g., a printer) or external devices such as one or more operator oruser stations 44, and an image server system 50 that are connected viathe network 42.

In one embodiment, computer executable instructions (e.g., programmedmodules and software) are stored in the memory 36 and/or data storagearea 38 and, when executed, enable the scanning system 11 to perform thevarious functions described herein. In this description, the term“computer readable storage medium” is used to refer to any media used tostore and provide computer executable instructions to the scanningsystem 11 for execution by the processor 245. Examples of these mediainclude data storage area 38 and any removable or external storagemedium (not shown) communicatively coupled with the scanning system 11either directly or indirectly, for example via the network 60.

FIG. 4 is a block diagram illustrating an example set of modules in thefluorescence scanner system 11 according to an embodiment of theinvention. In the illustrated embodiment, the modules include the tissuefinding module 305, autofocus module 310, auto exposure module 315,scanning workflow module 320, shading correction module 325, stripealignment module 330, data management module 335, and the visualizationand analysis module 340. In certain combinations, the variousillustrated modules collaborate to perform whole slide fluorescencescanning. These modules may be stored, for example, in memory 36 and/ordata storage 38, and executed by processors 20 or 245.

The tissue finding module 305 operates to determine the location of thesample 230 on the microscope slide. The location of the sample isdetermined with respect to the previously described XY coordinates forthe motorized stage. The tissue finding module 305 analyzes image datafrom the macro camera 240 to determine the location of the tissue.Advantageously, oblique illumination of the sample 230 results in themacro camera 240 providing a high contrast image of the sample 230 tothe tissue finding module 305. The tissue finding module 305 analyzesthe high contrast image from the macro camera 240 to determine thelocation of the sample 230. For example, the highest contrast areas maydefine the perimeter of the specimen to allow the tissue findingalgorithm 305 to determine an outline of the sample 230. In oneembodiment, the tissue finding module 305 employs a thresholdingalgorithm which, given an image largely composed of black pixels(representing areas where there is no tissue present) and white pixels(representing areas where there is tissue present), calculates theminimum pixel intensity of a white pixel (the “threshold”). Thethreshold is then used to classify each pixel in the macro image asbeing tissue or not tissue. This chosen threshold minimizes the resultof the following equation: ω_(nt)σ_(nt)+∫_(t)σ_(t)

where ω_(nt) is the probability that a pixel will be classified as nottissue, and σ_(nt) is the variance of intensities of pixels classifiedas not tissue, and ω_(t) is the probability that a pixel will beclassified as tissue, and σ_(t) is the variance of intensities of pixelsclassified as tissue.

The autofocus module 310 operates to generate a focus map of the surfaceof the sample 230 so that the digital slide image that is created as aresult of scanning the sample has optimal focus. The autofocus module310 initially determines a set of points on the sample using the XYcoordinates. Next, each point is visited by the TDI line scan camera,and the optimal focus height for the objective lens at each point isdetermined. In one embodiment, the optimal focus height for an XY pointcan be determined by scanning the XY point on the sample from the topend of the Z range of the objective to the bottom end of the Z range ofthe objective and then scanning the same point on the sample from thebottom end of the Z range of the objective to the top end of the Z rangeof the objective and then averaging the two. The height of the objectivethat provides the highest contrast in the scanned image data is thendetermined for both scans (i.e., top down and bottom up) and an averageheight is then calculated as the optimal focus height for that XY point.

In an alternative embodiment, the optimal focus height for an XY pointcan be determined by scanning the point on the sample from the top endof the Z range of the objective to the bottom end of the Z range of theobjective, or vice versa. The height of the objective that provides thehighest contrast in the scanned image data is then determined to be theoptimal focus height for that XY point.

The result of either technique for determining the optimal focus heightfor an XY point is a set of focus points that each comprise an XYlocation and a focus height. The set of focus points is then used tocalculate a non-planar focal surface that covers the scan area. The scanarea can include the entire sample 230 or just a portion of the sample230.

One particularly challenging aspect of determining the optimal focusheight for an XY point on the sample 230 arises from the use of the TDIline scan camera 205. This is because the sensor in a TDI line scancamera comprises a plurality of linear sensing arrays (e.g., 64 or 96)located side by side in a uniformly spaced, parallel manner. Duringrelative motion scanning, as the sample 230 moves perpendicular to thelinear sensing arrays, the sample 230 is sensed by the first array, thenthe second array, and so on. Advantageously, this provides multipleexposures of each area of the sample 230 as it moves relative to thesensor. The multiple exposures are then combined to create the resultingimage, which benefits from the increased exposure time withoutsacrificing the scanning speed. The increased exposure time isparticularly useful when used for faint fluorescence samples, asexplained above.

However, this advantage of the TDI line scan camera 205 becomes aliability when the perpendicular relative motion of the sample and thelinear sensing arrays is removed, as is the case when determining theoptimal focus height for an XY point on a sample. For example, as theTDI line scan camera 205 travels from the top end of its Z range to thebottom end of the Z range, each of the multiple linear sensing arrayscaptures image data for the portion of the sample 230 it can “see.” Asthe TDI camera 205 successively integrates the image data from thevarious linear sensing arrays, the image data is not from the same exactportion of the sample 230. Accordingly, when the image data from thevarious linear sensing arrays are combined to create a resulting image,the resulting image appears blurred. This is referred to as spatialblurring.

A related problem with using the TDI line scan camera 205 is referred toas temporal blurring. In temporal blurring, the problem is that as theobjective travels through its Z range, the first linear array sensorcaptures its image data at time t1. This image data is then integratedwith the image data captured by the second linear array sensor at timet2, and so on. By the time the 96^(th) linear array sensor is capturingits image at t96, the Z level of the objective has changed sufficientlyto be on a different focal plane. Thus, temporal blurring is also achallenge for identifying the optimal focus height for an XY point onthe sample 230.

Accordingly, the autofocus module 310 operates such that for each XYfocus point, the objective lens travels from the top end of its Z rangeto the bottom end of its Z range and then additionally travels back fromthe bottom end of its Z range to the top end of its Z range. The resultof the top-to-bottom scan and the bottom-to-top scan are then averagedto determine the optimal focus height. This effectively eliminates thetemporal blurring problems associated with using the TDI line scancamera 205 for identifying the optimal focus height at a focus point andallows the autofocus module 310 to use the average height of theobjective that provides the most contrast in the top-to-bottom scannedimage data and the bottom-to-top scanned image data as the optimal focusheight for each XY point.

In an alternative embodiment, to eliminate problems associated withtemporal blurring, during the top-to-bottom scan and during thebottom-to-top scan, the image data from each of the linear sensingarrays (e.g., from all 96 arrays) that is captured at each Z position isintegrated into a single line of data. In this fashion, there wouldstill be spatial blurring, but the temporal blurring is eliminated.

The autoexposure module 315 operates to determine the optimum exposuretime for each of the fluorochromes on a sample being scanned by thefluorescence scanning system 40. The exposure time determination processtypically uses a small area of the sample 230 that is representative ofthe overall sample 230 and the particular fluorochrome (i.e., theparticular channel). The autoexposure module 315 obtains focused imagedata from a small area of the sample 230 and analyzes the image data tocalculate an estimated optimal exposure time for the small area of thesample 230. The scan and analyze and calculate process is then repeateduntil the actual exposure time used by the TDI line scan camera 205 issubstantially equal to the estimated optimal exposure time that iscalculated.

Once the optimal exposure time has been calculated for one fluorochrome(i.e., channel) the autoexposure module 315 stores that exposure time ina data storage area for later use during the scan. The autoexposuremodule 315 then proceeds to determine the optimal exposure time for anyremaining fluorochromes on the sample 230. Advantageously, the scanningsystem 11 employs a monochrome TDI line scan camera 205 and separateexcitation and emission filter wheels, which allows the exposure timefor each fluorochrome to be calculated independent of the otherfluorochromes. This arrangement of the fluorescence scanning system 11provides a significant advantage over fluorescence scanning systems thatattempt to capture image data from multiple fluorochromes during onescanning movement.

The scanning workflow module 320 operates to manage the overall processfor creating a digital slide image of a fluorescence sample 230 usingthe fluorescence scanning system 40. As previously described, thescanning system 11 uses a monochrome TDI line scan camera 205 with highsensitivity and high bit depth to achieve optimum imaging performance.However, fluorescence samples 230 are typically marked with multiplefluorochromes, which causes light to be emitted from the sample 230 inmultiple wavelengths (i.e., channels). Accordingly, when scanning amulti-channel fluorescence sample 230, channel separation is achieved bythe fluorescence scanning system 11 by the use of specialized filters.For maximum flexibility, the excitation filter is mounted separatelyfrom the emission filter, as described above. This allows for multiplefilter combinations by means of a filter wheel having one or moreexcitation filters combined with a filter cube turret having one or moreemission filter cubes. Since the filter wheel is a motor controlleddevice, to minimize scanning time, it is preferable to also minimizefilter wheel rotations.

The scanning workflow module 320 can implement a very efficient processthat advantageously minimizes the number of filter wheel rotations. Forexample, compared to a conventional image tiling system, the scanningworkflow module 320 reduces the number of filter wheel rotations by afactor of 60 to 120. In a conventional image tiling system, for everysmall image tile, the filter wheel must be positioned “N” times, where Nequals the number of channels. For a typical exposure time of 10milliseconds per tile, significantly more time is spent rotating thefilter wheel N times than is spent actually sensing the images. Thefluorescence scanning system 40, in contrast, rotates the filter wheelonly N times for each scanned stripe. Since a typical scanned image tileis roughly 1 megapixel, whereas a typical scanned stripe is roughly 60megapixels, for each channel beyond the first, there is a 60 to 1decrease in the number of filter wheel rotations due to the efficiencyof the process implemented by the scanning workflow module 320.

The shading correction module 325 operates to correct for non-uniformityin the epifluorescence illumination optics and the TDI line scan camera205. The shading correction module 325 scans a small area of the sample320 (e.g., 1 mm) of the slide at a particular XY coordinate where nosample is present. In one embodiment, the scan is performed usingpredetermined focus parameters that were determined for the sample 320.The scan is performed at the maximum exposure time of the TDI line scancamera 205 in order to capture the light emitted by any residual dyepresent on the slide (background fluorescence). The average intensityfor each pixel column in the scan is calculated and checked to ensurethat an accurate illumination profile can be calculated, and then theshading correction module 325 calculates an illumination correctionprofile by comparing the average intensity of each pixel column to themaximum average intensity present in the image. This profile iscalculated for each fluorochrome (i.e., channel) to be scanned by thefluorescence scanning system 40.

The stripe alignment module 330 operates to align adjacent stripes ofimage data that are captured by the TDI line scan camera 205. In oneembodiment, the high precision XY accuracy of the motorized stage 255allows each stripe of image data to be abutted against its adjacentneighbor in the resulting single file digital slide image. The highprecision XY accuracy of the stage 255 therefore provides sufficientlyaligned adjacent stripes without the need for any software implementedalignment that is dependent upon an analysis of the content of the imagedata. This solves a particular problem with respect to software basedalignment of stripes of image data for fluorescence samples 230 thatarises because fluorescence sample image data typically does not containenough contrast in the overlap area of adjacent stripes to allowsoftware based alignment of stripes of fluorescence sample image data.

In an alternative embodiment, the scanning system 11 uses software basedalignment of stripes when there is sufficient contrast in thefluorescence sample image data. In this embodiment, the alignment ofadjacent stripes is not determined based on a certain number of pixelsdetermined to have the highest contrast in the overlap area of adjacentstripes. Instead, the stripe alignment module 330 calculates a contrastdistribution of the entire overlap area of the adjacent stripes. Thestripe alignment module 330 then identifies a contrast peak in thecontrast distribution of the overlap area and defines a band around thecontrast peak. The optimal stripe alignment is determined based on thepixels corresponding to the band around the contrast peak.Advantageously, oversaturated pixels are ignored when calculating thecontrast distribution.

Additionally, for multi-channel fluorescence samples 230, optimal stripealignment between adjacent stripes can be calculated for each channeland the channel providing the most robust alignment can be used.Advantageously, because the image data from the stripes corresponding tothe various channels are combined in the digital slide image, alignmentof only one channel between adjacent stripes is needed. Furthermore, thestripe alignment module 330 calculates stripe alignment in the directionperpendicular to the scanning direction one time. This can be calculatedbased on the high precision XY accuracy of the stage 255 in combinationwith the beginning of image data capture, which should be the same forall the channels.

The data management module 335 operates to manage the multi-channelimage data generated by the TDI line scan camera 205 and related imagedata and metadata information. Initially, as with bright-field digitalslide images, a fluorescence digital slide image scan can be stored in asingle digital slide image file. If the sample 230 was scanned atmultiple Z levels, the image for each of the various Z levels is alsoincorporated into the digital slide image file.

Additionally, because fluorescence scans typically include image datafrom multiple channels and each channel is related to the same sample230, it is advantageous to store the multi-channel image data a singledigital slide image file. Furthermore, it is also valuable to storerelated sub-imagery data and metadata related to instrument acquisitionsettings, image data descriptors, and sample information in the digitalslide image file. Moreover, any, known or scan-time computed inter-imagerelationships can also be stored in the digital slide image file.

Related sub-imagery may include an image of the slide label (e.g., abarcode), macro images of the whole slide, and a thumbnail of theacquired image. Metadata related to instrument acquisition settings mayinclude exposure time, filter specifications, light sourcespecifications, and calibration information. Metadata related to imagedata descriptors may include the intensity distribution of the imagepixels, automatically determined regions of interest and regions ofdisinterest, image features such as contrast distribution, frequencydistribution, texture, as well as figures of merit. Metadata related tosample information may include the tissue type and preparation as wellas targeted biologic feature. Inter-image relationships include imagetranslation and image rotation data.

In one embodiment, the fluorescence digital slide image file isstructured and stored as a tiled, multi-layer image. The base layer isat the original scan resolution and subsequent layers are sub-sampledresolutions that form an image pyramid. Each layer is made up of one ormore tiles and each tile of a layer can be compressed with a lossless ora lossy compression algorithm for improved disk and memory utilization,file transfer speeds and network image serving rates.

For multi-channel digital slide images, the base layer and eachsubsequent layer comprises the image data for each channel. For example,a four channel digital slide image would have a base layer divided intofour quadrants where each quadrant included a complete image of thesample 230 at one of the four quadrants.

The digital slide images are stored in a data storage area, for examplethe data storage area 38 of the scanning system 11 or the data storagearea 55 of an image server system 50 communicatively connected toscanning system 11 through network 42. In one embodiment, the datamanagement module 335 itemizes each scanned image with respect to itsassociated patient, specimen, and slide, and may also record results ofquantitative analysis in association with the stored digital slideimage. The data management module 335 may also provide a user withaccess to all of the stored information as well as provide an interfaceto hospital and laboratory information systems for data sharing.

In an alternative embodiment, separate digital slide images can becreated for each channel at which that the specimen 230 was scanned. Insuch an embodiment, a secondary file that references the related digitalslide image files is created. The secondary file comprises theinter-image relationships as well as visualization preferences andadjustments (described below). This secondary file is called a fusedimage.

The visualization and analysis module 340 operates to facilitate viewingand analysis of a fluorescence digital slide image file. Eachfluorescence digital slide image can be viewed at each of the variousseparate channels and/or viewed as a fused image where the image datafrom the various separate channels is overlayed into a single view ofthe sample that includes two or more channels. When the digital slideimage is viewed (in separate or fused channels) the image of the entiresample 230 is available for real time image navigation, rotation,zooming, magnification and Z level depth traversal.

In one embodiment, the multiple fluorochrome channels may be viewedsimultaneously by arranging them side by side or in a grid formation.Advantageously, the images are registered to enable synchronousnavigation. For example, a four channel scan may be arranged in a fourquadrant layout. In one embodiment, zooming one image causes all fourquadrants to similarly zoom. Additionally, panning left on one quadrant,for example, pans left on all four quadrants, and so on.

Image viewing adjustments such as brightness, contrast, gamma, and falsecoloring are automatically determined using the stored image descriptorsand the acquisition settings. In one embodiment, viewing adjustments canbe made by a user at a user station 44 for the individual images and/orfor a fused image (i.e., the combined image of two or more individualchannel images). In addition, when viewing a fused image the relativetranslation and rotation corrections may be adjusted.

Interactive image exploration tools are also enabled by the digitalvisualization and analysis module 340 to instantly access fluorochromeresponses on a cellular basis. Additionally, predetermined regions ofinterest may contain annotation that can be displayed to a user at theuser station 44 to indicate meaningful biologic responses or toautomatically quantitatively analyze. Additionally, the visualizationand analysis module 340 may provide a user at the user station 44 withtools to annotate regions of interest and then store such annotations inthe digital slide image file in relation to the base layer image.Advantageously, such annotations can be a useful to guide to documentartifacts in an image, regions of interest in an image, or to identify aregion of an image for reporting or quantitative analysis.

Additionally, the visualization and analysis module 340 may usepredetermined or otherwise identified image features to locate similarimage data or patterns using content based image retrieval techniques.Advantageously, this utility can provide a user at the user station 44with related case information and image data.

In one embodiment, a client-server architecture permits a user at theuser station 44 to view a fluorescence digital slide image located atthe image server system 50 or the scanning system 11 by requesting thecompressed image tiles at a specified pyramid level on an as neededbasis and by performing client-side caching of tiles in anticipation ofuser requests.

The digital visualization and analysis module 340 additionally operatesto facilitate whole slide quantitative analysis of the fluorescencedigital slide images, whether the image is a quadrant style image or afused style image. In one embodiment, the digital visualization andanalysis module 340 can facilitate a quantitative analysis of aparticular region of interest, instead of the entire digital slideimage. Analysis results can be stored in a data storage area such asdata storage areas 38, 55, or a data storage area of operator or userstations 44 for use with data management and reporting.

FIG. 5 is a block diagram illustrating an example wired or wirelessprocessor enabled system that may be used in connection with variousembodiments described herein. For example, the system 550 may be used inconjunction with the scanning devices illustrated in FIGS. 1-3. As willbe clear to those skilled in the art, alternative processor enabledsystems and/or architectures may also be used.

The system 550 preferably includes one or more processors, such asprocessor 560. Additional processors may be provided, such as anauxiliary processor to manage input/output, an auxiliary processor toperform floating point mathematical operations, a special-purposemicroprocessor having an architecture suitable for fast execution ofsignal processing algorithms (e.g., digital signal processor), a slaveprocessor subordinate to the main processing system (e.g., back-endprocessor), an additional microprocessor or controller for dual ormultiple processor systems, or a coprocessor. Such auxiliary processorsmay be discrete processors or may be integrated with the processor 560.

The processor 560 is preferably connected to a communication bus 555.The communication bus 555 may include a data channel for facilitatinginformation transfer between storage and other peripheral components ofthe system 550. The communication bus 555 further may provide a set ofsignals used for communication with the processor 560, including a databus, address bus, and control bus (not shown). The communication bus 555may comprise any standard or non-standard bus architecture such as, forexample, bus architectures compliant with industry standard architecture(“ISA”), extended industry standard architecture (“EISA”), Micro ChannelArchitecture (“MCA”), peripheral component interconnect (“PCI”) localbus, or standards promulgated by the Institute of Electrical andElectronics Engineers (“IEEE”) including IEEE 488 general-purposeinterface bus (“GPIB”), IEEE 696/S-100, and the like.

System 550 preferably includes a main memory 565 and may also include asecondary memory 570. The main memory 565 provides storage ofinstructions and data for programs executing on the processor 560. Themain memory 565 is typically semiconductor-based memory such as dynamicrandom access memory (“DRAM”) and/or static random access memory(“SRAM”). Other semiconductor-based memory types include, for example,synchronous dynamic random access memory (“SDRAM”), Rambus dynamicrandom access memory (“RDRAM”), ferroelectric random access memory(“FRAM”), and the like, including read only memory (“ROM”).

The secondary memory 570 may optionally include a internal memory 575and/or a removable medium 580, for example a floppy disk drive, amagnetic tape drive, a compact disc (“CD”) drive, a digital versatiledisc (“DVD”) drive, etc. The removable medium 580 is read from and/orwritten to in a well-known manner. Removable storage medium 580 may be,for example, a floppy disk, magnetic tape, CD, DVD, SD card, etc.

The removable storage medium 580 is a non-transitory computer readablemedium having stored thereon computer executable code (i.e., software)and/or data. The computer software or data stored on the removablestorage medium 580 is read into the system 550 for execution by theprocessor 560.

In alternative embodiments, secondary memory 570 may include othersimilar means for allowing computer programs or other data orinstructions to be loaded into the system 550. Such means may include,for example, an external storage medium 595 and an interface 570.Examples of external storage medium 595 may include an external harddisk drive or an external optical drive, or and external magneto-opticaldrive.

Other examples of secondary memory 570 may include semiconductor-basedmemory such as programmable read-only memory (“PROM”), erasableprogrammable read-only memory (“EPROM”), electrically erasable read-onlymemory (“EEPROM”), or flash memory (block oriented memory similar toEEPROM). Also included are any other removable storage media 580 andcommunication interface 590, which allow software and data to betransferred from an external medium 595 to the system 550.

System 550 may also include a communication interface 590. Thecommunication interface 590 allows software and data to be transferredbetween system 550 and external devices (e.g. printers), networks, orinformation sources. For example, computer software or executable codemay be transferred to system 550 from a network server via communicationinterface 590. Examples of communication interface 590 include a modem,a network interface card (“NIC”), a wireless data card, a communicationsport, a PCMCIA slot and card, an infrared interface, and an IEEE 1394fire-wire, just to name a few.

Communication interface 590 preferably implements industry promulgatedprotocol standards, such as Ethernet IEEE 802 standards, Fiber Channel,digital subscriber line (“DSL”), asynchronous digital subscriber line(“ADSL”), frame relay, asynchronous transfer mode (“ATM”), integrateddigital services network (“ISDN”), personal communications services(“PCS”), transmission control protocol/Internet protocol (“TCP/IP”),serial line Internet protocol/point to point protocol (“SLIP/PPP”), andso on, but may also implement customized or non-standard interfaceprotocols as well.

Software and data transferred via communication interface 590 aregenerally in the form of electrical communication signals 605. Thesesignals 605 are preferably provided to communication interface 590 via acommunication channel 600. In one embodiment, the communication channel600 may be a wired or wireless network, or any variety of othercommunication links. Communication channel 600 carries signals 605 andcan be implemented using a variety of wired or wireless communicationmeans including wire or cable, fiber optics, conventional phone line,cellular phone link, wireless data communication link, radio frequency(“RF”) link, or infrared link, just to name a few.

Computer executable code (i.e., computer programs or software) is storedin the main memory 565 and/or the secondary memory 570. Computerprograms can also be received via communication interface 590 and storedin the main memory 565 and/or the secondary memory 570. Such computerprograms, when executed, enable the system 550 to perform the variousfunctions of the present invention as previously described.

In this description, the term “computer readable medium” is used torefer to any non-transitory computer readable storage media used toprovide computer executable code (e.g., software and computer programs)to the system 550. Examples of these media include main memory 565,secondary memory 570 (including internal memory 575, removable medium580, and external storage medium 595), and any peripheral devicecommunicatively coupled with communication interface 590 (including anetwork information server or other network device). Thesenon-transitory computer readable mediums are means for providingexecutable code, programming instructions, and software to the system550.

In an embodiment that is implemented using software, the software may bestored on a computer readable medium and loaded into the system 550 byway of removable medium 580, I/O interface 585, or communicationinterface 590. In such an embodiment, the software is loaded into thesystem 550 in the form of electrical communication signals 605. Thesoftware, when executed by the processor 560, preferably causes theprocessor 560 to perform the inventive features and functions previouslydescribed herein.

The system 550 also includes optional wireless communication componentsthat facilitate wireless communication over a voice and over a datanetwork. The wireless communication components comprise an antennasystem 610, a radio system 615 and a baseband system 620. In the system550, radio frequency (“RF”) signals are transmitted and received overthe air by the antenna system 610 under the management of the radiosystem 615.

In one embodiment, the antenna system 610 may comprise one or moreantennae and one or more multiplexers (not shown) that perform aswitching function to provide the antenna system 610 with transmit andreceive signal paths. In the receive path, received RF signals can becoupled from a multiplexer to a low noise amplifier (not shown) thatamplifies the received RF signal and sends the amplified signal to theradio system 615.

In alternative embodiments, the radio system 615 may comprise one ormore radios that are configured to communicate over various frequencies.In one embodiment, the radio system 615 may combine a demodulator (notshown) and modulator (not shown) in one integrated circuit (“IC”). Thedemodulator and modulator can also be separate components. In theincoming path, the demodulator strips away the RF carrier signal leavinga baseband receive audio signal, which is sent from the radio system 615to the baseband system 620.

If the received signal contains audio information, then baseband system620 decodes the signal and converts it to an analog signal. Then thesignal is amplified and sent to a speaker. The baseband system 620 alsoreceives analog audio signals from a microphone. These analog audiosignals are converted to digital signals and encoded by the basebandsystem 620. The baseband system 620 also codes the digital signals fortransmission and generates a baseband transmit audio signal that isrouted to the modulator portion of the radio system 615. The modulatormixes the baseband transmit audio signal with an RF carrier signalgenerating an RF transmit signal that is routed to the antenna systemand may pass through a power amplifier (not shown). The power amplifieramplifies the RF transmit signal and routes it to the antenna system 610where the signal is switched to the antenna port for transmission.

The baseband system 620 is also communicatively coupled with theprocessor 560. The central processing unit 560 has access to datastorage areas 565 and 570. The central processing unit 560 is preferablyconfigured to execute instructions (i.e., computer programs or software)that can be stored in the memory 565 or the secondary memory 570.Computer programs can also be received from the baseband processor 610and stored in the data storage area 565 or in secondary memory 570, orexecuted upon receipt. Such computer programs, when executed, enable thesystem 550 to perform the various functions of the present invention aspreviously described. For example, data storage areas 565 may includevarious software modules (not shown) that were previously described withrespect to FIGS. 2 and 3.

Various embodiments may also be implemented primarily in hardware using,for example, components such as application specific integrated circuits(“ASICs”), or field programmable gate arrays (“FPGAs”). Implementationof a hardware state machine capable of performing the functionsdescribed herein will also be apparent to those skilled in the relevantart. Various embodiments may also be implemented using a combination ofboth hardware and software.

Furthermore, those of skill in the art will appreciate that the variousillustrative logical blocks, modules, circuits, and method stepsdescribed in connection with the above described figures and theembodiments disclosed herein can often be implemented as electronichardware, computer software, or combinations of both. To clearlyillustrate this interchangeability of hardware and software, variousillustrative components, blocks, modules, circuits, and steps have beendescribed above generally in terms of their functionality. Whether suchfunctionality is implemented as hardware or software depends upon theparticular application and design constraints imposed on the overallsystem. Skilled persons can implement the described functionality invarying ways for each particular application, but such implementationdecisions should not be interpreted as causing a departure from thescope of the invention. In addition, the grouping of functions within amodule, block, circuit or step is for ease of description. Specificfunctions or steps can be moved from one module, block or circuit toanother without departing from the invention.

Moreover, the various illustrative logical blocks, modules, and methodsdescribed in connection with the embodiments disclosed herein can beimplemented or performed with a general purpose processor, a digitalsignal processor (“DSP”), an ASIC, FPGA or other programmable logicdevice, discrete gate or transistor logic, discrete hardware components,or any combination thereof designed to perform the functions describedherein. A general-purpose processor can be a microprocessor, but in thealternative, the processor can be any processor, controller,microcontroller, or state machine. A processor can also be implementedas a combination of computing devices, for example, a combination of aDSP and a microprocessor, a plurality of microprocessors, one or moremicroprocessors in conjunction with a DSP core, or any other suchconfiguration.

Additionally, the steps of a method or algorithm described in connectionwith the embodiments disclosed herein can be embodied directly inhardware, in a software module executed by a processor, or in acombination of the two. A software module can reside in RAM memory,flash memory, ROM memory, EPROM memory, EEPROM memory, registers, harddisk, a removable disk, a CD-ROM, or any other form of storage mediumincluding a network storage medium. An exemplary storage medium can becoupled to the processor such the processor can read information from,and write information to, the storage medium. In the alternative, thestorage medium can be integral to the processor. The processor and thestorage medium can also reside in an ASIC.

The above description of the disclosed embodiments is provided to enableany person skilled in the art to make or use the invention. Variousmodifications to these embodiments will be readily apparent to thoseskilled in the art, and the generic principles described herein can beapplied to other embodiments without departing from the spirit or scopeof the invention. Thus, it is to be understood that the description anddrawings presented herein represent a presently preferred embodiment ofthe invention and are therefore representative of the subject matterwhich is broadly contemplated by the present invention. It is furtherunderstood that the scope of the present invention fully encompassesother embodiments that may become obvious to those skilled in the artand that the scope of the present invention is accordingly not limited.

The invention claimed is:
 1. A method for standardizing a fluorescencemicroscopy imaging system, the method comprising: by a first imagingsystem to be standardized, capturing a drift image comprising an imageof a drift reference slide; calculating a drift measurement using thedrift image; by the first imaging system, capturing a firstnormalization image comprising an image of a normalization slide,wherein the normalization slide comprises a specimen which is preparedwith a fluorochrome; by a reference imaging system, capturing areference normalization image comprising an image of the normalizationslide; comparing the first normalization image to the referencenormalization image; and determining a gamma value and offset value forthe first imaging system based on the comparison.
 2. The method of claim1, wherein capturing a first normalization image, comparing the firstnormalization image to the reference normalization image, anddetermining a gamma value and offset value for the first imaging systemare all performed for each of a plurality of normalization slides,wherein each of the plurality of normalization slides comprises aspecimen which is prepared with a different fluorochrome.
 3. A methodfor standardizing a fluorescence microscopy imaging system, the methodcomprising: by a first imaging system to be standardized, capturing adrift image comprising an image of a drift reference slide; calculatinga drift measurement using the drift image; by the first imaging system,capturing a first normalization image comprising an image of anormalization slide; by a reference imaging system, capturing areference normalization image comprising an image of the normalizationslide; comparing the first normalization image to the referencenormalization image, wherein comparing the first normalization image tothe reference normalization image comprises calculating a firsthistogram based on the first normalization image, calculating areference histogram based on the reference normalization image, andcomparing the first histogram to the reference histogram; anddetermining a gamma value and offset value for the first imaging systembased on the comparison.
 4. The method of claim 3, wherein comparing thefirst normalization image to the reference normalization image furthercomprises: identifying a reference area in the reference normalizationimage; calculating a root-mean-squared difference of each of one or morecandidate areas of the first normalization image to the reference area;and identifying one of the one or more candidate areas having the lowestroot-mean-squared difference to the reference area.
 5. The method ofclaim 3, wherein comparing the first histogram to the referencehistogram comprises: calculating a reference cumulative distributionfunction for the reference histogram; calculating a first cumulativedistribution function for the first histogram; and determining one ormore corresponding intensities in the first normalization image and thereference normalization image for which the first cumulativedistribution function and the reference cumulative distribution functionmatch.
 6. The method of claim 5, wherein determining a gamma value andoffset value for the first imaging system comprises: determining a gammafunction based on the one or more corresponding intensities; andapplying a linear regression to the gamma function to generate a linearequation comprising the gamma value and the offset value.
 7. The methodof claim 6, further comprising estimating a standard error in the linearregression.
 8. A system for standardizing a fluorescence microscopyimaging system, the system comprising: at least one hardware processor;and at least one executable module that, when executed by the at leastone hardware processor, receives a drift image comprising an image of adrift reference slide captured by a first imaging system, calculates adrift measurement using the drift image, receives a first normalizationimage comprising an image of a normalization slide captured by the firstimaging system, receives a reference normalization image comprising animage of the normalization slide captured by a reference imaging system,compares the first normalization image to the reference normalizationimage, wherein comparing the first normalization image to the referencenormalization image comprises calculating a first histogram based on thefirst normalization image, calculating a reference histogram based onthe reference normalization image, and comparing the first histogram tothe reference histogram; and determines a gamma value and offset valuefor the first imaging system based on the comparison.
 9. The system ofclaim 8, wherein comparing the first normalization image to thereference normalization image further comprises: identifying a referencearea in the reference normalization image; calculating aroot-mean-squared difference of each of one or more candidate areas ofthe first normalization image to the reference area; and identifying oneof the one or more candidate areas having the lowest root-mean-squareddifference to the reference area.
 10. The system of claim 8, whereincomparing the first histogram to the reference histogram comprises:calculating a reference cumulative distribution function for thereference histogram; calculating a first cumulative distributionfunction for the first histogram; and determining one or morecorresponding intensities in the first normalization image and thereference normalization image for which the first cumulativedistribution function and the reference cumulative distribution functionmatch.
 11. The system of claim 10, wherein determining a gamma value andoffset value for the first imaging system comprises: determining a gammafunction based on the one or more corresponding intensities; andapplying a linear regression to the gamma function to generate a linearequation comprising the gamma value and the offset value.